Tutorial 4 Introduction - membranes.
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Transcript of Tutorial 4 Introduction - membranes.
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Tutorial 4
Introduction - membranes
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What are membranes?Membranes are barriers that define compartments
• They are made up of a lipid bilayer
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Membrane Proteins
• They carry out many functions
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Integral - firmly anchored into the membrane by hydrophobic interactions with the hydrophobic portion of the bilayer.
Membrane Proteins
Peripheral - attached to surface of membrane and to the exposed parts of integral proteins. Held in place by polar interactions primarily.
Transmembrane proteins - extend through membrane
Lipid anchored proteins - have covalently attached lipid molecules that anchor the protein into the bilayer
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Freeze Fracture• A technique used to visualize protein
distribution in a membrane
This figure was modified from Bloom and Fawcett, A Textbook of Histology, Chapman and Hall, N.Y., Twelfth Edition, 1994, Figure 1-3
Is the exposed surface made of polar or nonpolar groups?
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Amino Acid Number
50 100 150 200 250
0
Ave
rage
Hyd
ropa
thy
Inde
xHydropathy Plots
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Hyd
roph
obic
ity
0
Hydropathy plot for Glycophorin A
How many times does this protein span the membrane?
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Membrane Experiments #1:
Gel Electrophoresis can be used to identify the types of proteins found within a membrane.
• Different stains can tell you different things about the membrane proteins
• Different cell preparations can allow you to view internal vs external proteins
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Experiment• Gel 1 – stain ALL proteins with Coomassie Blue• Gel 2 – remove Coomassie Blue and stain with a
carbohydrate stain.• Gel 3 – Treat fresh cells with a fluorescent marker
that binds proteins on the outside of cells.• Gel 4 – Using fresh cells, make inside-out vesicles.
Label inside out vesicles using a fluorescent marker.
• Gel 5 – Separate free proteins from the membranes.
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A
DE
Gel 3
A
BC
Gel 4
B
D
Gel 5
A
BCDE
A
E
Gel 1 Gel 2All Carbohydrates outside inside Free proteins
OUTSIDE
INSIDE
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• Polypeptide A:Found both on inside and outside of membrane. How many times does it span the membrane???
We can solve this with a hydropathy plot!
0
What can we conclude from these results?
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Membrane Experiments #2:
MEMBRANE FLUIDITY can be observed using fluorescent dyes.
- Different dyes can label different proteins- Proteins can move laterally quite easily
and this can be observed
Note: only certain proteins are able to flip to the other membrane bilayer via a flippase and this is quite specialized.
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- Surface proteins of cultured cells are labeled with antibodies coupled to fluorescent dyes (red and green).
- The "red" and "green" cells are then mixed and can fuse.
- In time, labeled proteins from each cell mix showing membrane fluidity
CELL FUSION
Figure 11-35 (p.366)
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An experiment to demonstrate fluidity of membrane components. The green indicates green fluorescence
FRAP – fluorescence recovery after photobleaching
A = the location of the spot to be photobleached B = the spot after being bleached by radiationC, D = disappearance of the spot as time goes on
due to the fluidity of the membrane
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NEXT TUTORIAL:
STRUCTURE AND FUNCTION OF MITOCHONDRIA AND
CHLOROPLAST!